Abnormally low amounts of the cell cycle inhibitor p27 due to excessive proteolysis are hallmarks of several types of aggressive tumors. It has been shown that low levels of p27 expression are independent prognostic markers in many human cancers correlating with higher tumor grade and poor survival. The abundance of p27 is cell cycle regulated and controlled primarily by a proteasome-mediated degradation pathway (3). Degradation of p27 in G1 and S phase is mediated by the SCFSkp2 ubiquitin ligase and requires the activity of Cksl. Consistent with this view was the finding that an elevated level of Skp2 was detected in several types of human tumors. Skp2 levels correlate with grade of malignancy and decreased p27 expression. Ubiquitination and degradation of p27 require direct physical interactions between Skp2 and Cksl. The mechanisms by which Cksl promotes p27 degradation and its functional relevance to human cancers have not been rigorously investigated. The objective of this proposal is to define the precise molecular mechanisms by which Skp2 interacts with Cksl to catalyze p27 ubiquitination. The central hypothesis of this proposal is that interactions between Skp2 and Cksl are mediated by a few surface exposed amino acids. Binding of Cksl to Skp2 creates a docking site for phosphorylated p27 to enter the E3 ubiquitin ligase complex. Disruption of the Cks1-Skp2 interaction may result in stabilization p27 and enhanced cellular responses to antiproliferative signals. This hypothesis is based on our preliminary results of identification of critical amino acid residues in both Skp2 and Cksl that are involved in their association and ubiquitination of p27. We plan to test our hypothesis by pursuing the following specific aims: (a) Identification and characterization of amino acid residues in Skp2 that are required for association with Cksl and ubiquitination of p27. (2) Determine the molecular mechanism by which Cks1 stimulates ubiquitination of phosphorylated p27 by SCFSkp2. (3) Determine whether disruption of the Cks1-Skp2 interaction in vivo stabilizes p27 and enhances cellular responses to antiproliferative signals. Understanding how normal cells regulate proper levels of p27 and the mechanism responsible for abnormally low levels of p27 in tumor cells will reveal potential targets for therapeutic intervention.